Cysteinate Protonation and Water Hydrogen Bonding at the Active

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Cysteinate Protonation and Water Hydrogen Bonding at the ActiveSite of a Nickel Superoxide Dismutase Metallopeptide-Based Mimic: Implications for the Mechanism of Superoxide Reduction Jason Shearer,* Kristy L. Peck, Jennifer C. Schmitt, and Kosh P. Neupane Department of Chemistry, University of Nevada, Reno, Reno, Nevada 89557, United States S Supporting Information *

ABSTRACT: Nickel-containing superoxide dismutase (NiSOD) is a mononuclear cysteinate-ligated nickel metalloenzyme that catalyzes the disproportionation of superoxide into dioxygen and hydrogen peroxide by cycling between NiII and NiIII oxidation states. All of the ligating residues to nickel are found within the first six residues from the N-terminus, which has prompted several research groups to generate NiSOD metallopeptide-based mimics derived from the first several residues of the NiSOD sequence. To assess the viability of using these metallopeptide-based mimics (NiSOD maquettes) to probe the mechanism of SOD catalysis facilitated by NiSOD, we computationally explored the initial step of the O2− reduction mechanism catalyzed by the NiSOD maquette {NiII(SODm1)} (SODm1 = HCDLP CGVYD PA). Herein we use spectroscopic (S K-edge X-ray absorption spectroscopy, electronic absorption spectroscopy, and circular dichroism spectroscopy) and computational techniques to derive the detailed active-site structure of {NiII(SODm1)}. These studies suggest that the {NiII(SODm1)} active-site possesses a NiII-S(H+)-Cys(6) moiety and at least one associated water molecule contained in a hydrogen-bonding interaction to the coordinated Cys(2) and Cys(6) sulfur atoms. A computationally derived mechanism for O2− reduction using the formulated active-site structure of {NiII(SODm1)} suggests that O2− reduction takes place through an apparent initial outersphere hydrogen atom transfer (HAT) from the NiII-S(H+)-Cys(6) moiety to the O2− molecule. It is proposed that the water molecule aids in driving the reaction forward by lowering the NiII-S(H+)-Cys(6) pKa. Such a mechanism is not possible in NiSOD itself for structural reasons. These results therefore strongly suggest that maquettes derived from the primary sequence of NiSOD are mechanistically distinct from NiSOD itself despite the similarities in the structure and physical properties of the metalloenzyme vs the NiSOD metallopeptide-based models. Superoxide (O2−) is a toxic reactive oxygen species (ROS) formed through the adventitious one electron reduction of dioxygen.1,2 As it is an unavoidable byproduct of aerobic respiration organisms have developed pathways to degrade O2− before it can initiate extensive cellular damage.2−7 The most prevalent O2− degradation enzymes are the superoxide dismutases (SODs).2−6 These enzymes utilize redox active transition-metal cofactors to catalyze superoxide disproportionation through a highly efficient ping-pong type mechanism by accessing reduced (SODred) and oxidized (SODox) redox states: SODred + O−2 + 2H+ → SODox + H 2O2

(1a)

SODox + O2− → SODred + O2

(1b)

structure (the so-called nickel binding hook).13−15 The nickel binding hook has the consensus sequence HCXXPC and contains all of the residues required for nickel coordination.11 In the reduced form of NiSOD, the NiII ion is contained within a square-planar NiN2S2 coordination environment with ligands derived form the His(1) N-terminal amine nitrogen, the amidate nitrogen from Cys(2), and cysteinate sulfur atoms derived from Cys(2) and Cys(6) (Scheme 1). Oxidation to the NiIII oxidation state yields a square-pyramidal coordination geometry about nickel following the coordination of the His(1) imidazole δ-nitrogen atom. O2− approaches the Ni-center from Scheme 1

The most recently discovered SOD is nickel-containing superoxide dismutase (NiSOD), which is widely expressed by aquatic and soil bacteria.4,5,8−12 As its name suggests, NiSOD contains a redox-active nickel cofactor at its active site and facilitates superoxide disproportionation by cycling between reduced NiII and oxidized NiIII oxidation states. In its active form NiSOD is homohexameric with each monomer containing a mononuclear nickel ion. The nickel ion is situated at the bottom of a ∼15 Å long channel within an N-terminal loop © XXXX American Chemical Society

Received: August 3, 2014

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dx.doi.org/10.1021/ja5079514 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

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thioether complexes, 2 8 which was suggestive that {NiII(SODm1H(1)HMe)} contained a NiII-S(H+)-Cys moiety. Variable pH studies supported this notion; at pH 9.5, where the NiII-S(H+)-Cys moiety would become deprotonated, the preedge feature corresponding to the thiolate S(1s) → Ψ1* transition in the S K-edge X-ray absorption spectrum of {NiII(SODm1H(1)HMe)} appeared. Solomon and co-workers have demonstrated that reduced NiSOD likely contains a NiIIS(H+)-Cys moiety as well.28 Furthermore, metric parameters and spectroscopic signatures of reduced NiSOD and the maquettes are similar to one another. Taken together this suggests similar reduced active-site structures for the metalloenzyme vs the NiSOD maquette. In this study we further probe the O2− reduction mechanism facilitated by these NiSOD maquettes. Herein we present the preparation of an S-methylated derivative of {NiII(SODm1)}, {NiII(SODm1C(6)CMe)} (CMe = S-methylcysteine; Chart 1). {NiII(SODm1C(6)CMe)} was utilized to aid in identifying the cysteinate sulfur atom that comprises the NiII-S(H+)-Cys moiety within the parent metallopeptide. To better quantify the active-site structures of both {NiII(SODm1C(6)CMe)} and {NiII(SODm1)}, detailed spectroscopic and computational studies of {NiII(SODm1C(6)CMe)} and {NiII(SODm1)} under high and low pH were undertaken. These results yielded a detailed active-site structure of the catalytically active form of {Ni I I (SOD m 1 )}. The resulting structure of active {NiII(SODm1)} was then used to provide accurate computational models to examine the mechanism of O2− reduction facilitated by the metallopeptide. The results presented in this study strongly suggest that, although NiSOD and {NiII(SODm1)} likely have similar active-site structures, the mechanisms by which the two systems facilitate SOD catalysis are distinct.

the surface through the 15 Å channel associating near the Ni center trans to the imidazole ligand. We, and others, have utilized metallopeptides-based mimics derived from the NiSOD sequence (NiSOD maquettes) to explore the chemistry of NiSOD itself.12,16−24 These NiSOD maquettes have been shown to replicate the geometric structure, electronic structure, and spectroscopic properties of NiSOD. Furthermore, these NiSOD maquettes are catalytically active, displaying high SOD activity with kcat values approaching 6 × 108 M−1 s−1 for some metallopeptide-based models.12 We have utilized a family of NiSOD maquettes derived from the first 12 residues of the Streptomyces coelicolor NiSOD sequence, the {Ni(SODm1)} family (SODm1 = HCDLPCGVYD-PA; Chart 1).16−18,20,21 These SOD active metalChart 1. Active Site Structures of the Metallopeptides from the {Ni(SODm1)} Family

lopeptides have proven useful in correlating different functional and structural aspects of NiSOD with its sequence. In one study the mechanism of O2− disproportionation catalyzed by {Ni(SODm1H(1)HMe)} was probed.16 {Ni(SODm1H(1)HMe)} is a derivative of the parent {Ni(SODm1)} metallopeptide that contains an N-methyl histidine (HMe) in place of His(1) (SODm1H(1)HMe = HMeCDLP-CGVYD-PA). In that study O2− reduction facilitated by reduced {NiII(SODm1H(1)HMe)} was proposed to proceed through a proton-coupled electrontransfer (PCET) reaction with a significant quantum mechanical tunneling component. This was evidenced by a large room temperature solvent H/D kinetic isotope effect (KIE, ∼20) and the resulting Arrhenius activation parameters (virtually barrierless reaction). Considering the similarities in the reduced structures and disproportionation kinetics of {Ni I I (SOD m 1 H(1)H M e )} vs other members of the {NiII(SODm1)} family it is a reasonable supposition that the O2− reduction mechanism occurs via a nearly identical process among the {NiII(SODm1)} family. Spectroscopic studies suggested that the transferred “H•” (proton and electron) within {NiII(SODm1H(1)HMe)} originates from a protonated coordinated NiII-S(H+)-Cys moiety. High vs low pH S K-edge X-ray absorption spectroscopy performed on {NiII(SODm1H(1)HMe)} was most insightful in formulating the NiII-S(H+)-Cys moiety. In transition-metal complexes containing at least one hole in the nd-set a prominent peak in the pre-edge region of the S K-edge X-ray absorption corresponding to a thiolate S(1s) → Ψ1* transition is typically observed (Ψ1* corresponds to the lowest-energy acceptor state(s), which is typically metal nd in character).25−27 The S K-edge X-ray absorption spectrum of {NiII(SODm1H(1)HMe)} obtained at pH 8.0 displayed no prominent pre-edge feature corresponding to a thiolate S(1s) → Ψ1* transition. Furthermore, the edge was significantly broader than is typically observed for NiII-thiolates. The pH 8.0 S K-edge X-ray absorption spectrum obtained for {NiII(SODm1H(1)HMe)} was in fact similar to what would be predicted for NiII-

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RESULTS AND ANALYSIS S-Methylcysteine Peptide Metalation and Nickel Binding. To approximate the influence of a protonated coordinated cysteinate ligand on the active-site of {NiII(SODm1)}, a series of Cys methylated derivatives of SODm1 were prepared. Attempts to coordinate NiII to either the Cys(2)CysMe (H2N-HCMeDLP-CGVYD-PA-COOH; CysMe = S-methylcysteine) or Cys(2/6)Cys Me (H 2 N-HC Me DLPCMeGVYD-PA-COOH) derivatives were unsuccessful. Over the pH range of 7.0 to 9.0 no evidence for NiII incorporation into the peptides was noted. In contrast, NiII will readily bind to the Cys(6)CysMe derivative (SODm1C(6)CMe; H2N-HCDLP-CMeGVYD-PACOOH) forming {Ni II(SOD m1 C(6)C Me)}. Solutions of {NiII(SODm1C(6)CMe)} in N-ethylmorpholine (NEM) buffer (50 mM NEM; pH 7.4) are reminiscent of the pH 7.4 solution of {NiII(SODm1)}. Solutions of both metallopeptides are light pinkish beige in color.17 However, unlike solutions of {NiII(SODm1)}, which display a color change as the pH is elevated (pinkish beige to reddish brown),18 solutions of {NiII(SODm1C(6)CMe)} do not change color over the pH range of 7.0−9.5. We also note that unlike {NiII(SODm1)},17 {NiII(SODm1C(6)CMe)} displayed no apparent redox activity over the range of −0.2 to 1.0 V vs SHE as determined by cyclic voltammetry and attempts to trap a NiIII species following chemical oxidation. It therefore appears that the NiIII oxidation state is not accessible over the range relevant for SOD catalysis. Thus, it is not surprising that {NiII(SODm1C(6)CMe)} is SOD inactive. B



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The pH 7.4 NiII binding constant to SODm1C(6)CMe was determined spectrophotometrically using glycine as a competitive NiII chelating agent. These titrations yielded a NiII Kd value from SODm1C(6)CMe of 3(1) μM at pH 7.4. This NiII dissociation constant compares well with other Kd values we have previously measured for NiSOD maquettes.17,19 Thus, the Cys(6)CysMe modification is not having a major impact on NiII binding to the peptide in comparison to the parent unmodified peptide. The geometry about the NiII center of {NiII(SODm1C(6)Me C )} was probed by Ni K-edge X-ray absorption spectroscopy (Figure 1). The XANES region of the Ni K-edge X-ray

tional models for the active-sites of these metallopeptides were generated by approximating the reduced NiSOD active-site as a Ni(II) center coordinated to a 2-amino-N-(2-mercaptoethyl)acetamidate ligand and an ethanethiolate coordinated to nickel trans to the amidate nitrogen ((NiII(SOD))−). The ethanethiolate was then either methylated, generating NiII(SODCys(6)Me), or protonated, generating NiII(SOD-Cys(6)H+).31 In addition, a computational model was constructed with a water molecule hydrogen bonded to the cysteinate sulfur atoms (NiII(SODCys(6)H+)···H2O). Geometry optimized structures were obtained using closed-shell restricted density functional theory (DFT, S = 0) using the BP86 functional with the def2tzvp basis set and the COSMO solvation model with parameters applicable to water (Figure 2, Table 1).32 We note that NiII(SOD-Cys(6)H+) is thermodynamically more stable than Cys(2) protonated NiII(SOD-Cys(2)H+) (ΔG = −4.5 kcal mol−1). The computationally derived structure of unprotonated (Ni(SOD))− displays Ni−S bond lengths of 2.178 (transamine) and 2.235 Å (trans-amidate) and Ni−N bond lengths of 1.972 (amine) and 1.889 Å (amidate). Although the average Ni−N bond length is consistent with the structural data for both {NiII(SODm1)} (pH 7.4) and NiSOD itself, the average calculated Ni−S bond length is longer than the experimentally determined Ni−S bond lengths for these systems (2.21 Å (calcd) vs 2.18 Å (exp.), Table 1). Instead, the average calculated Ni−S bond length matches the EXAFS derived average Ni−S bond length for {NiII(SODm1H(1)HMe)} previously recorded at pH 9.5.16 The Cys(6) S-methylated computational model NiII(SODCys(6)Me) possesses a Ni(II)−Sthiolate bond length of 2.172 Å, a Ni(II)−Sthioether bond length of 2.208 Å, a Ni(II)−Namine bond length of 1.996 Å, and a Ni(II)−Namide bond length of 1.864 Å (Table 1). The resulting average Ni−S and Ni−N bond lengths calculated for NiII(SOD-Cys(6)Me) therefore match those obtained experimentally for {NiII(SODm1C(6)CMe)}. An important structural aspect of Cys(6) methylation revealed by these computational models is the contraction of the Ni−S bond trans to the amide in NiII(SOD-Cys(6)Me) relative to (NiII(SOD))− by 0.027 Å. This is due to the relief of repulsive filled/filled Ni(3dπ)−S(3p) interactions between the metal center and the ligand upon methylation.33−36 Not only does the trans-amidate Ni−S bond contract upon methylation of the thiolate ligand, but it also contracts upon protonation relative to the trans-amidate Ni−S bond found in (NiII(SOD))−. Upon protonation of the Cys(6) sulfur atom the Ni−S bond contracts by 0.03 Å relative to (NiII(SOD))− (2.205 Å) without effecting a dramatic change on the other Ni−S bond length. Furthermore, the average Ni−S bond length obtained for NiII(SODCys(6)H+) (2.19 Å) more closely matches the experimentally derived average pH 7.4 Ni−S bond length obtained for {NiII(SODm1)} (2.18 Å).

Figure 1. Nickel K-edge X-ray absorption spectrum of {NiII(SODm1C(6)CMe)}. The left spectrum depicts the XANES region of the Ni Kedge X-ray absorption spectrum. The top right spectrum depicts the unfiltered k3 EXAFS spectrum (red spectrum), the simulation to the data (blue spectrum), and the difference spectrum between the experimental and simulated spectrum (green dashed spectrum). The bottom right spectrum depicts the magnitude Fourier transformed k3 EXAFS spectrum (red spectrum), the simulation to the data (blue spectrum), and the difference spectrum between the experimental and simulated spectrum (green dashed spectrum). Simulation parameters: Eo = 8341.2 eV; shell no. 1: Ni−S, n = 2, r = 2.194(5) Å, σ = 0.006(1) Å 2; shell no. 2: Ni−N, n = 2, r = 1.913(3) Å, σ = 0.006(1) Å 2; ε2 = 1.08.

absorption spectrum displays a small feature at 8331.8(2) eV (area = 0.02(1) eV relative to the edge) corresponding to the Ni(1s → 3d) transition and a more intense shoulder at 8337.7(1) eV (area = 0.41(1) eV relative to the edge) corresponding to the Ni(1s → 4pz) transition. Both of these features are consistent with square-planar NiII.29 The EXAFS data are consistent with a four coordinate NiN2S2 complex. The best fit to the {NiII(SODm1C(6)CMe)} EXAFS data yielded two Ni−N scatterers at 1.91 Å and two Ni−S scatterer at 2.19 Å (Chart 1, Table 1).30 Computational Models for {NiII(SODm1C(6)CMe)} and {NiII(SODm1)} in Different Protonation States. Computa-

Table 1. Selected Bond Lengths (in Å) for Computational Models (BP86/def2-tzvp), {NiII(SODm1)} (Ni K-edge EXAFS), and {NiII(SODm1C(6)CMe)} (Ni K-edge EXAFS) NiII(SOD-C(6)CMe) b

Ni−S(1) Ni−S(2)d Ni−N(1)e Ni−N(2)f a

2.172 2.208 1.996 1.864

NiII(SOD-C(6)H+) 2.174 2.205 1.995 1.858

NiII(SOD-C(6)H+)···H2O 2.175 2.190 1.992 1.862

(NiII(SOD))− 2.178 2.235 1.972 1.889

{NiII(SODm1)} pH 7.4a c

2.18 − 1.91c −

{NiII(SODm1C(6)CMe)} 2.19c − 1.91c −

Refs 17 and 20. btrans-amine. cEXAFS derived average Ni−S bond length. dtrans-amidate. eAmine nitrogen atom. fAmidate nitrogen atom. C



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Figure 2. Geometry optimized models (top; BP86/def2-tzvp) and HOMO−3 through LUMO (PBE0/def2-tzvp) for (NiII(SOD))−, NiII(SODCys(6)Me), NiII(SOD-Cys(6)H+), and NiII(SOD-Cys(6)H+)···H2O.

doubly occupied HOMO and HOMO−1 are S(3π*)-Ni(3dπ) and S(3π)-Ni(3dπ) antibonding orbitals. Next in energy is the Ni(3dz2)-S(3σ) HOMO−2 followed by the Ni(3dxy)+S(3p)Ni(2p) HOMO−3. NiII(SOD-Cys(6)Me) and NiII(SOD-Cys(6)H+) are electronically similar to one another. Upon protonation or methylation the Ni(3d x2 −y 2 ) LUMO and the Ni(3d z 2 ) HOMO−2 remain relatively unchanged compared to the MOs for (NiII(SOD))− with perturbations to their absolute energies and the Ni-ligand character to the MOs, which reflects the differential bonding in the three computational models. These perturbations include a reduction in ligand character and an increase in Ni character to the LUMO of NiII(SODCys(6)H+/Me) relative to (NiII(SOD))−, which reflects the reduction in ligand-Ni covalency to this MO. The HOMO−2, in contrast, contains more ligand character from the amine and amide moieties. Furthermore, although the HOMO−2 remains primarily Ni(3dz2) in character, it now contains significant contributions from the Ni(3dxy) orbital (the Ni(3d) orbital is ∼60% Ni(3dz2) and ∼40% Ni(3dxy)). In addition, the amide oxygen and nitrogen atomic orbitals that participate in bonding are rotated in the HOMO−2 for NiII(SOD-Cys(6)H+/Me) relative to (NiII(SOD))−. In (NiII(SOD))− the amide oxygen is perpendicular to the xy plane resulting in a π-type interaction with the Ni(3d) orbital, while in NiII(SOD-Cys(6)H+/Me) the orbitals are contained within the xy plane resulting in a σ-type interaction with the Ni(3d) orbital. More substantial changes observed for these two computational models with respect to (NiII(SOD))− are found in the doubly filled orbitals resulting from Ni(3dπ)−S(3π) interactions (the HOMO, HOMO−1, and HOMO−3). This is a result of the unavailability of the Cys(6)-S(3px) orbital for π-bonding to the nickel center upon protonation or methylation. The HOMO of NiII(SOD-

Addition of a water molecule contained in a hydrogenbonding interaction to the cysteinates (NiII(SODCys(6)H+)··· H2O) yielded a further shortening of the Ni−S bond trans to the amidate nitrogen, resulting in an average Ni−S bond length of 2.18 Å. The average Ni−S bond length matches the previously reported EXAFS data for {NiII(SODm1)} at pH 7.4. Furthermore, the Ni ligand bond lengths also match the EXAFS37 and crystallographically14,15 derived bond lengths of NiSOD itself. We note that the addition of a water molecule also reorients the S−H+ moiety such that the proton is positioned within the Cys(6)S−Ni−Namine cleft. This structural aspect will become important for the proposed O2− reduction mechanism. The average Ni−S bond lengths of NiII(SODCys(2)H+) and NiII(SODCys(2)H+)···H2O (2.21 and 2.20 Å) are long relative to the EXAFS data obtained for {NiII(SODm1)}. This is due to an elongated Ni−S bond trans to the amidate (2.247 Å) that is not substantially shortened upon Cys(6)S···H2O hydrogen bonding. Taken together these data seem to suggest that at pH 7.4 {NiII(SODm1)} possesses a Cys(6) sulfur atom that is protonated and that there is likely at least one water molecule associated hydrogen bonded to the Cys(2) and Cys(6) sulfur atoms. This formulation is supported by S K-edge X-ray absorption spectroscopy (see next section). Isosurface plots for the LUMO through HOMO−3 for NiII(SOD-Cys(6)Me), NiII(SOD-Cys(6)H+), NiII(SOD-Cys(6)H+)···H2O, and (NiII(SOD))− are displayed in Figure 2. The atomic orbital populations for the LUMO+1 through HOMO−4 for these computational models are displayed in Table 2 (PBE0/def2-tzvp). The LUMO through HOMO−3 calculated for (NiII(SOD))− are in line with what has been previously reported.18,39 The unoccupied LUMO is best described as a Ni(3dx2−y2)-N/S(σ*) antibonding orbital. The D



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Table 2. Orbital Energy (normalized to the HOMO) and Atomic Orbitals Composition of Cys(6) Methylated, Cys(6) and Cys(2) Protonated, and Unprotonated NiSOD Computational Modelsa orbital

(NiII(SOD))−

NiII(SOD-C(6)CMe)

NiII(SOD-C(6)H+)

NiII(SOD-C(6)H+)···H2O

LUMO+1

energy: 5.08 eV Ni 3d: 6.1% S(a) 3p: 2.1% S(b) 3p: 0.3% N(a) 2p: 1.2% N(b) 2p: 2.1% O 2p: 0.8% energy: 4.45 eV Ni 3d: 41.7% S(a) 3p: 16.4% S(b) 3p: 28.2% N(a) 2p: 3.3% N(b) 2p: 2.6% O 2p: 0.4% energy: 0 eV Ni 3d: 19.5% S(a) 3p: 16.4% S(b) 3p: 52.1% N(a) 2p: 0.4% N(b) 2p: 0.3% O 2p: 0.2% energy: −0.17 eV Ni 3d: 26.0% S(a) 3p: 40.4% S(b) 3p: 1.5% N(a) 2p: 1.8% N(b) 2p: 0.5% O 2p: 2.6% energy: −0.84 eV Ni 3d: 75.2% S(a) 3p: 2.3% S(b) 3p: 8.0% N(a) 2p: 2.2% N(b) 2p: 0.5% O 2p: 1.1% energy: −0.90 eV Ni 3d: 35.2% S(a) 3p: 18.8% S(b) 3p: 3.7% N(a) 2p: 14.2% N(b) 2p: 1.1% O 2p: 9.5% energy: −1.43 eV Ni 3d: 6.8% S(a) 3p: 2.1% S(b) 3p: 0.3% N(a) 2p: 1.2% N(b) 2p: 3.5% O 2p: 0.8%




LUMO

HOMO

HOMO−1

HOMO−2

HOMO−3

HOMO−4

a

All orbital compositions were derived from a Lowden population analysis of the PBE0/def2-tzvp single point calculations. S(a) trans-amine; S(b) trans-amide; N(a) amide; N(b) amine.

Cys(6)Me/H+) becomes primarily S/N(π)-Ni(3dπ) antibonding in character. Similarly, the HOMO−3 becomes a predominantly S/N(π)-Ni(3dxy/3dz2) antibonding orbital. The one major difference between the two computational models is the HOMO−1. For NiII(SOD-Cys(6)Me) the HOMO−1 is derived from a Cys(2)S/Namide(π)−Ni(3dπ) antibonding interaction, while the HOMO−1 for NiII(SOD-Cys(6)H+) is

derived from a Cys(6)S/Namine/Namide (σ)−Ni(3dxz+3dxy) antibonding interaction. Upon the addition of the water molecule to the Cys(6) protonated model (NiII(SOD-Cys(6)H+)···H2O), the LUMO and HOMO remain relatively unchanged compared with waterfree NiII(SOD-Cys(6)H+), save an increase in S-character to the LUMO. This increase in covalency to the LUMO is due to E



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spectra of {NiII(SODm1)}. For most transition-metal thiolate compounds with one or more holes in the nd-set the S K-edge X-ray absorption spectrum contains a pre-edge feature that is resolved from the edge. This peak corresponds to a S(1s) → Ψ1* transition, where Ψ1* is the lowest-energy acceptor state. For these metallopeptides Ψ1* is predominately Ni(3dx2−y2) in character. As S K-edge XAS is an atomic spectroscopy the acceptor state must contain sulfur character. Therefore, the S(1s) → Ψ1* transition gains intensity through the mixing of S(3p) character into the acceptor state. Thus, integration of the pre-edge feature provides a direct measurement of metalthiolate covalency in such compounds. The integrated intensity of the S(1s) → Ψ1* transition, IS(1s)→Ψ1*, can be related to the degree of S(3p) character in the acceptor state by

the contraction of the Cys(6) Ni−S bond upon protonation, which enhance S−Ni σ-bonding in the xy plane. We also note that there appears to be electronic communication through the S···H2O hydrogen bonds as there is a small amount of electron density residing on the water O atom in the HOMO; the HOMO contains ∼1.2% water O(2pz/y) character. Other influences on the electronic structure are also induced upon water molecule hydrogen bonding. Owing to protonation and the hydrogen-bonding of water to Cys(6), the HOMO−1 becomes similar to the HOMO−1 of NiII(SOD-Cys(6)Me); it is best described as a Cys(2)S/Namide(π)-Ni(3dπ) antibonding orbital. There is also a small contribution from the water O(2pz/y) atomic orbitals to the HOMO−1 (0.03 Å. An indication of Cys(2)/Cys(6) water association is the contraction of the average Ni−S bond lengths in the calculated model NiII(SODCys(6)H+)···H2O such that it matches the pH 7.4 EXAFS derived Ni−S bond lengths for {NiII(SODm1)}. A similar influence on Ni−S bond lengths upon their interaction with hydrogen-bond donors was observed by Harrop in a small molecule NiSOD mimic.34 We note that the difference in the average calculated bond lengths for NiII(SOD-Cys(6)H+) and NiII(SOD-Cys(6)H+)···H2O is 0.014 Å, which could relate to inadequacies in the computational methods used coupled with the error in the EXAFS derived bond lengths of {NiII(SODm1)}. However, S K-edge spectroscopy strongly suggests that there is indeed a water molecule associated with the active-site of {NiII(SODm1)} at pH 7.4. The S K-edge spectroscopic data for the high pH form of {NiII(SODm1)} can be readily rationalized in terms of the structure outlined in Chart 2 (no Cys protonation and no water hydrogen bonding to the active-site). The S K-edge spectrum yields the expected thiolate S(1s) → Ψ1* transition with covalency parameters that correlate well with the computationally expected values. The electronic absorption and CD spectra of {NiII(SODm1)} obtained at high pH are also fully consistent with the active-site formulation presented in Chart 2. Similarly, {NiII(SODm1C(6)CMe)} yields fully consistent S K-edge and

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DISCUSSION NiSOD maquettes derived from the NiSOD protein sequence have similar spectroscopic, reactive, and structural properties as the metalloenzyme.12,16−24 A number of detailed studies have been performed using these maquettes to better understand the properties and mechanism of NiSOD itself. One major unresolved question is how closely related the NiSOD maquettes’ reactivity and mechanism of catalysis are to NiSOD itself.12 A recent study by us demonstrated that a NiSOD maquette, {NiII(SODm1H(1)HMe)}, facilitated O2− reduction through a probable PCET mechanism.16 This was based largely on kinetic (KIE ∼20 at room temperature) and thermodynamic (pKa and redox potential) arguments. Considering the similarities between {NiII(SODm1H(1)HMe)} and other metallopeptides derived from the NiSOD sequence, such as {NiII(SODm1)}, it was deemed a reasonable supposition that all of these metallopeptides facilitate O2− disproportionation through a similar mechanism. Therefore, the parent metallopeptide {NiII(SODm1)} was subjected to detailed studies to better define the details of its active-site. This information was then utilized to examine a viable mechanism for O2− reduction computationally, which can in-turn be used to make conclusions concerning the relationship between the superoxide disproportionation mechanism effected by {NiII(SODm1)} peptides vs NiSOD. J



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{NiII(SODm1)} catalyzed reduction of O2−. The first step of the superoxide reduction mechanisms for both water-free Cys(6) protonated Ni II (SOD-Cys(6)H + ) and water associated NiII(SOD-Cys(6)H+)···H2O was considered. We deemed an outersphere process for the reduction of O2− by {NiII(SODm1)} most likely based on the fact that, with the exception of stoichiometric amounts of cyanide, no exogenous ligands have been shown to be capable of binding to the NiII center of NiSOD,39 {NiII(SODm1)},48 other NiSOD maquettes19 or the vast majority of amidate-containing NiIIN2S2 complexes with one notable exception (vide inf ra).44,49 These exogenous ligands include halides and superoxide-like mimics such as N3− and NO•. In addition to electronic factors, we suspect that exogenous ligands may be incapable of binding to the nickel center of NiSOD maquettes for steric reasons. {NiII(SODm1)}, and other similar maquettes, lacks the outersphere hydrogen bonds found in NiSOD that may be important in maintaining an open loop-like structure that would allow superoxide to access the nickel center. DFT geometry optimizations performed on a peptide-based computational model comprised of the first six residues of the NiSOD N-terminus shows that the peptide-loop and histidine imidazole group effectively block O2− from accessing the NiII center (Figure 9). In contrast to the

optical spectra with an active-site formulation of a methylated Cys(6) sulfur atom and no water association at the active-site. Computational studies demonstrate that Cys(6) protonation vs methylation of {NiII(SODm1)} yield virtually identical electronic structures and spectroscopic signatures demonstrating that {NiII(SODm1C(6)CMe)} can be used as a mimic of Cys(6) protonated {NiII(SODm1)}. However, {NiII(SODm1C(6)CMe)} is spectroscopically and structurally distinct from {NiII(SODm1)} under low pH conditions, suggesting that simple protonation of Cys(6) alone cannot be used to rationalize the available data. It is only upon the addition of a water molecule contained in a hydrogen-bonding network to Cys(2) and Cys(6) that the S K-edge XAS and optical spectra can be fully rationalized. The lack of a well-resolved thiolate S(1s) → Ψ1* transition can be rationalized by the proposal hydrogen bonding to the Cys(2) thiolate effectively blue-shifts the transition into the edge. In fact, this thiolate S(1s) → Ψ1* transition is observed as a shoulder feature in the edge of the experimental low pH S K-edge spectrum of {NiII(SODm1)}. In a study examining water hydrogen bonding to Ni and Fe thiolates, Solomon and co-workers found that the S K-edge XAS pre-edge transition of a bis-amine NiIIN2S2 complex (Ni(DACO)) shifted by +0.2 eV upon water hydrogen bonding in dry acetonitrile.47 Water association in the NiII(SODCys(6)H+)···H2O computational model yields a Ni−S bond contraction relative to (NiII(SOD))− and an increase in the S(3p) contribution to the LUMO, neither of which are seen in Ni(DACO) upon water hydrogen bonding. Thus, a larger shift of the thiolate S(1s) → Ψ1* transition energy is produced in NiII(SOD-Cys(6)H+)···H2O than observed in Ni(DACO). All of the spectroscopic data for the low pH form of {NiII(SODm1)} can be rationalized using a computational model containing only one water molecule associated with the cysteinate sulfur atoms within the primary hydration sphere. It was deemed possible that additional important water−active site interactions are present in the low pH form of {NiII(SODm1)}. When an additional water molecule is placed about the thiolate S atoms in the NiII(SOD-Cys(6)H+)···H2O computational model, it does not associate directly with the primary coordination sphere (Supporting Information). Instead, the additional water molecule hydrogen bonds to the associated water molecule (i.e., it would be associated with the hydration sphere). This imparts no significant change to the electronic or geometric structure of the nickel center. Thus, the active-site structures presented in Chart 2 likely represent the important interactions that contribute to the properties of {NiII(SODm1)}. The differences in active-site water association between the active-sites probed (i.e., {NiII(SODm1C(6)CMe)} vs the high and low pH forms of {NiII(SODm1)}) may be due to differences in the peptide environment about the Ni center in the metallopeptides. Owing to the likely Cys(6) methyl group orientation, {NiII(SODm1C(6)CMe)} possesses a more hydrophobic S−NiII−S cleft where water would hydrogen bond to the cysteinates. The methyl group also yields steric crowding about the already congested water hydrogen-bonding site as well. Both of these may prevent water association to Cys(2) and Cys(6). Changes in peptide conformation at high vs low pH may also prevent water from associating at the active-site of the high pH form of {NiII(SODm1)} due to steric crowding. Mechanistic Insight Into the Superoxide Reduction Half Reaction Facilitated by {NiII(SODm1)}. In this study we computationally probed an outersphere mechanism for the

Figure 9. Space filling models of two different orientations of the geometry optimized structure (BP86/def2-tzvp) for the peptide-loop structure of {NiII(SODm1)} looking down the loop (right) and at the Ni(II)-S(H+)-H2N cleft (left).

sterically restricted NiII center of {NiII(SODm1)}, the H2N− Ni(II)-Cys(6)H+ cleft is well exposed to solvent making it accessible for O2− attack (Figure 9). This is the location where we propose O2− abstracts a formal H• from the {NiII(SODm1)} active site. A similarly crowded nickel center was formulated using NMR spectroscopic studies performed by Tietze and co-workers on an 8-mer NiSOD maquette.24 In that study a CN− anion was proposed to be associated within the active-site, bound directly to Ni in a highly unusual η2 fashion as opposed to the typically observed end-on fashion through the cyanide C atom. If correct, this binding mode is likely necessitated by the steric crowding imposed by the peptide loop folding over the nickel center. The CN− ligand is thus prevented from coordinating to NiII in the more typical linear end-on fashion. We also note that Tietze’s study indicated that a cysteinate S-hydrogen-bonded water molecule was likely present at the nickel active-site, further supporting our active-site formulation for {NiII(SODm1)}. The computational investigations disclosed above seem to suggest a reaction mechanism that may be described as a HATlike reaction. Although these studies suggest that a HAT-like reaction can occur from the site lacking the Cys(2)/Cys(6)··· water hydrogen-bonding interaction (i.e., Ni(SOD-Cys(6))*), it is not as facile as when this interaction is present; the K



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activation barrier for H• transfer is over 10 kcal mol−1 higher in NiII(SOD-Cys(6)H+)* vs NiII(SOD-Cys(6)H+)···H2O. Decomposition of the thermodynamic properties related to concerted proton- and electron-transfer (PT−ET) reactions (i.e., pKa and NiII/NiIII Eox) suggests that the lowered reaction barrier in NiII(SOD-Cys(6)H+)* vs NiII(SOD-Cys(6)H+)··· H2O results from the increased acidity of the Cys-S(H+)-NiII proton upon water hydrogen bonding to Cys(6). In contrast to the redox potentials, which remain virtually unaltered,50 the Cys-S(H+)-NiII proton in NiII(SOD-Cys(6)H+)···H2O has a pKa three pKa units lower than NiII(SOD-Cys(6)H+). From a simple application of Marcus−Hush theory the higher acidity of the Ni II(SOD-Cys(6)H +)···H2 O proton relative to the NiII(SOD-Cys(6)H+)* proton will destabilize the NiII(SODCys(6)H+)···H2O reactant state, which will in turn reduce the minimum crossing point energy between the reactant and product PESs, thus resulting in a lowered activation barrier. This computationally proposed mechanism is consistent with a mechanistic study published by us probing the O 2 − disproportionation kinetics/thermodynamics facilitated by {NiII(SODm1H(1)HMe)}.16 First, the thermodynamic parameter obtained experimentally suggested that {NiII(SODm1H(1)HMe)} is poised to reduce O2− through a PCET reaction. Equilibrium experiments between {NiII/III(SODm1H(1)HMe)} and TEMPO•/TEMPO-H placed the bond dissociation free energy (BDFE) for the abstractable H• within {NiII(SODm1H(1)HMe)} at 75 kcal mol−1. Application of a modified Bordwell equation51 to the experimentally derived pKa and Eox values for {NiII(SODm1H(1)HMe)} (pKa ∼ 8.4; Eox ∼ 0.44 V vs SHE) yielded a NiII-S(H+)Cys BDFE of 79 kcal mol−1. Both are less than the peroxide HO2− BDFE (81 kcal mol−1),51 thus {NiII(SODm1H(1)HMe)} is poised to reduce O2− to HO2− despite the fact that {NiII(SODm1H(1)HMe)} is neither thermodynamically capable of protonating (HO2 pKa = 4.69)1 nor reducing superoxide (high pH O2− Ered = +0.20 V)1 independent of the other process. We note that the computationally derived pKa, E1/2, and NiII-S(H+)Cys BDFEs for NiII(SOD-Cys(6)H+)* (80.1 kcal mol−1) and NiII(SODCys(6)H+)···H2O (75.6 kcal mol−1) are in line with the experimentally derived values reported for {NiII(SODm1H(1)HMe)}. The other computationally derived thermodynamic properties for the O2− reduction reaction are also in line with the experimental data. On the basis of the {NiII(SODm1H(1)HMe)} H• BDFE it was speculated that the ΔG for the {NiII(SODm1H(1)HMe)} facilitated O2− reduction reaction is between −3 and −10 kcal mol−1. The computationally derived Gibbs free energies for O2− reduction facilitated by NiII(SODCys(6)H+)* (ΔG = −10.9 kcal mol−1) and NiII(SODCys(6)H+)···H2O (ΔG = −11.7 kcal mol−1) are therefore consistent with the experimentally derived ΔG for O2− reduction by these nickel centers. The computationally derived observation that O2− reduction may proceed through a HAT process is also consistent with the kinetic data obtained for O2− reduction facilitated by {NiII(SODm1H(1)HMe)}. Superoxide disproportionation catalyzed by {NiII(SODm1H(1)HMe)} proceeds with a large room temperature solvent H/D KIE of ∼20. This strongly suggested: (a) H• movement in the rate limiting step and (b) a quantum mechanical tunneling component in the reaction mechanism. Our computational results support the notion of H• movement in the rate-limiting step of the superoxide reduction halfreaction facilitated by the {Ni(SODm1)} family.

The proposed mechanism is based solely on an examination of the orbitals involved in ET and PT along the doublet PES.32 It has been noted that it is difficult to distinguish between HAT vs PCET vs consecutive PT/ET reactions on the basis of MO arguments alone.52,53 The fact that minimal charge transfer to superoxide is observed at the TS for the O2− reduction reaction facilitated by NiII(SOD-Cys(6)H+)···H2O may indicate that the PT−ET mechanism is not as simple as outlined above. Therefore, a study probing the more detailed aspects of the proposed reaction mechanism, such as adiabaticity and quantum mechanical tunneling, is forthcoming. Biological Relevance of {NiII(SODm1)} to NiSOD. Considering the similarities in the spectroscopic and structural data between these NiSOD maquettes and NiSOD itself, it is probable that NiSOD possesses a NiII-S(H+)-Cys(6) moiety and additional hydrogen bonds to the coordinated cysteinate sulfur atoms. Alternatively, multiple hydrogen bonds to Cys(6) and a NiII-S(H+)-Cys(2) moiety might also be envisioned. These hydrogen bonds and the protonated NiII-cysteinate are likely responsible for the observed spectroscopic and geometric properties of reduced NiSOD, which cannot be adequately reproduced in their absence. Functionally, these hydrogen bonds and cysteinate protonation likely serve two purposes. One is to fine-tune the physical properties of the nickel center for optimal SOD catalysis. Another likely role is protection of the cysteinate sulfurs from oxidative damage induced by O2 and H2O2. We, and others, have commented on the robustness of the NiSOD active site toward ROSs.12,21,34,40,42,54−56 The Grapperhaus research group and our research group have suggested this is due (in part) to the electronic consequences of the mixed amine/amidate ligand set.21,40,42,54 Harrop and Grapperhaus have also suggested that hydrogen bonds to the cysteinate sulfurs will protect the cysteinates from oxidative damage induced by ROSs.34,54 This study shows that {NiII(SODm1)} contains both a NiII-S(H+)-Cys(6) moiety and water cysteinate hydrogen bonds, which likely enhance its observed robustness against oxidative damage in addition to the electronic finetuning of the Ni−S bond imparted by the primary coordination environment. A similar protective mechanism is likely occurring in NiSOD itself. Concerning the proposed mechanism for O2− reduction by {NiII(SODm1)}, there are several lines of evidence pointing to the fact that the proposed mechanism is not valid for NiSOD itself. The first comes from the observation that the kinetics of NiSOD catalysis is virtually pH independent.37,57 This shows that proton movement is not rate-limiting in the enzymatic process and strongly argues against a concerted ET-PT process. A second line of evidence that the above mechanism is not biologically valid comes from the NiSOD structure itself.13−15 An approach of the O2− molecule toward the proton in the H2N−Ni(II)-Cys(6)H+ cleft as proposed for {NiII(SODm1)} is impossible to achieve in the metalloenzyme. Instead, the only access point of superoxide to the nickel center involves O2− traveling down a protein channel ending in the open nickelbinding loop structure. Outersphere ET from the Ni(II) center of NiSOD to O2− as superoxide approaches the active-site could then occur. It is also possible that an inner-sphere mechanism is occurring in the metalloenzyme itself. Such an innersphere process was considered by Pelmenschikov and Siegbahn in a hybrid DFT study probing the NiSOD mechanism.58 In that study an innersphere mechanism was demonstrated to be L



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ACKNOWLEDGMENTS This work was supported by the National Science Foundation (CHE-1362662). All X-ray absorption studies were performed at the NSLS. Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-98CH10886. Ni K-edge studies were preformed on beamline X3-b, which is supported through the Case Center for Synchrotron Biosciences, which is funded through the National Institute of Biomedical Imaging and Bioengineering (NIH grant no. P30-EB-009998)

feasible. In addition to an inner- vs outersphere process, Pelmenschikov’s mechanism was distinct from ours in that (a) the NiII center was considered to be in the S = 1, not S = 0 spinstate; (b) the mechanism is best described as an ET process followed by a PT process; and (c) Cys(2), not Cys(6), was considered as the proton donor. The proposal that Cys(2) is the likely proton donor is because the Cys(6) sulfur atom will not effectively position the H+ toward the active-site and therefore makes for a less effective PT process to the coordinated O22− than that facilitated by Cys(2). A recent study by Masuda and co-workers lends further support to a possible innersphere superoxide disproportionation mechanism.44 In their study a small molecule mixed amine/amidate square-planar NiN2S2 complex facilitated superoxide reduction and oxidation. In the absence of protons or an axial ligand the coordinated superoxide behaves as a pseduohalide. Reduction of superoxide within the purported NiII−O2− adduct was only effected by the addition of both protons and an axial imidazole ligand to the nickel center. Such a mechanism is unlikely to occur in {NiII(SODm1)}. Owing to steric constraints O2− can only coordinate to {NiII(SODm1)} in the same position where the axial histidine imidazole would coordinate. Additional axial ligation to the vacant site of the five coordinate superoxide adduct would be precluded for steric reasons. Because superoxide reduction appears to necessitate the formation of a six-coordinate NiII species, O2− reduction would not occur. We also note that unlike Pelmenschikov and Siegbahn’s mechanism, the proton source proposed by Masuda was the coordinated amine N−H group, negating the need for the direct involvement of the Ni(II)-S(H+)-Cys group in catalysis. If true the positioning of the Ni(II)-S(H+)-Cys proton toward the active-site may be irrelevant for catalysis negating the need for Cys(2) protonation in the mechanism. In summary, NiSOD maquettes derived from the primary NiSOD sequence remain useful mimics for understanding the electronic and geometric structure of NiSOD. However, considering the above observations, any disproportionation mechanism operable in NiSOD itself would have very little relationship to the O2− disproportionation mechanism of these maquettes. Thus, direct insight into the enzymatic disproportionation mechanism gained from such mimics may be tenuous at best.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Contains experimental details, Cartesian coordinates for all computational models discussed, calculated electronic absorption spectra, SODm1C(6)CMe glycine competitive Ni(II) binding assay results, and spectroscopic parameters obtained from the Gaussian deconvolution of the experimental CD and electronic absorption spectra of {NiII(SODm1)} (pH 7.4 and 9.5) and {NiII(SODm1)}. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

[email protected] Notes

The authors declare no competing financial interest. M



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(51) Warren, J. J.; Tronic, T. A.; Mayer, J. M. Chem. Rev. 2010, 110, 6961−7001. (52) Hammes-Schiffer, S.; Stuchebrukhov, A. Chem. Rev. 2010, 110, 6939−6960. (53) Layfield, J. P.; Hammes-Schiffer, S. Chem. Rev. 2014, 114, 3466−3494. (54) Mullins, C. S.; Grapperhaus, C. A.; Kozlowski, P. M. J. Biol. Inorg. Chem. 2006, 11, 617−625. (55) Herdt, D. R.; Grapperhaus, C. A. Dalton Trans. 2012, 41, 364− 366. (56) Green, K. N.; Brothers, S. M.; Jenkins, R. M.; Carson, C. E.; Grapperhaus, C. A.; Darensbourg, M. Y. Inorg. Chem. 2007, 46, 7536− 7544. (57) Bryngelson, P. A.; Arobo, S. E.; Pinkham, J. L.; Cabelli, D. E.; Maroney, M. J. J. Am. Chem. Soc. 2004, 126, 460−461. (58) Pelmenschikov, V.; Siegbahn, P. E. M. J. Am. Chem. Soc. 2006, 128, 7466−7475.

(28) Szilagyi, R. K.; Bryngelson, P. A.; Maroney, M. J.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 3018− 3019. (29) Colpas, G. J.; Maroney, M. J.; Bagyinka, C.; Kumar, M.; willis, W. S.; Suib, S. L.; Mascharack, P. K.; Baidya, N. Inorg. Chem. 1991, 30, 920−928. (30) The EXAFS data can be modeled such that the Ni-−S shell is divided into two scatterers with one Ni−S scatterer at 2.17 Å and one at 2.21 Å (Supporting Information). However, these shells are to close for them to be resolved based on the quality of the data presented (Δr = π/(2Δk) with Δk = 13 Å−1). (31) In these computational models Cys(6) will refer to the thiolate sulfur atom trans to the amidate nitrogen atom, and Cys(2) will refer to the thiolate sulfur atom trans to the amine nitrogen atom for ease of reference to the NiSOD and {NiII(SODm1)} active-site. (32) NEVPT2 calculations using a CAS(3,6) active space suggest that there is minimal multiconfigurational character to the ground-state wave function suggesting single determinant methods are sufficient to describe these systems. (33) Gale, E. W.; Patra, A. K.; Harrop, T. C. Inorg. Chem. 2009, 48, 5620−5622. (34) Gale, E. M.; Narendrapurapu, B. S.; Simmonett, A. C.; Schaefer, H. F., III; Harrop, T. C. Inorg. Chem. 2010, 49, 7080−7096. (35) Grapperhaus, C. A.; Darensbourg, M. Y. Acc. Chem. Res. 1998, 31, 451−459. (36) Farmer, P. J.; Reibenspies, J. H.; Lindahl, P. A.; Darensbourg, M. Y. J. Am. Chem. Soc. 1993, 115, 4665. (37) Choudhury, S. B.; Lee, J.-W.; Davidson, G.; Yim, Y.-I.; Bose, K.; Sharma, M. L.; Kang, S.-O.; Cabelli, D. E.; Maroney, M. J. Biochemistry 1999, 38, 3744−3752. (38) There is a potential error associated with these values as they are derived from a mixture of computational (at the PBE0/def2-tzvp level) and experimental values; see Supporting Information for full details. (39) Fiedler, A. T.; Bryngelson, P. A.; Maroney, M. J.; Brunold, T. C. J. Am. Chem. Soc. 2005, 127, 5449−5462. (40) Shearer, J.; Dehestani, A.; Abanda, F. Inorg. Chem. 2008, 47, 2649−2660. (41) DeBeer George, S.; Neese, F. Inorg. Chem. 2010, 49, 1849− 1853. (42) Shearer, J.; Zhao, N. Inorg. Chem. 2013, 45, 9637−9639. (43) Mathrubootham, V.; Thomas, J.; Staples, R.; McCraken, J.; Shearer, J.; Hegg, E. L. Inorg. Chem. 2010, 49, 5393−5406. (44) Nakane, D.; Wasada-Tsutsui, Y.; Funahashi, Y.; Hatanaka, T.; Ozawa, T.; Masuda, H. Inorg. Chem. 2014, 53, 6512−6523. (45) Kruger, H. J.; Peng, G.; Holm, R. H. Inorg. Chem. 1991, 30, 734−742. (46) Mayer, J. M.; Hrovat, D. A.; Thomas, J. L.; Borden, W. T. J. Am. Chem. Soc. 2002, 124, 11142−11147. (47) Dey, A.; Green, K. N.; Jenkins, R. M.; Jeffrey, S. P.; Darensbourg, M.; Hodgson, K. O.; Hedman, B.; Solomon, E. I. Inorg. Chem. 2007, 46, 9655−9660. (48) In this study we have probed the ability of {NiII(SODm1)} to coordinate N3−, Cl−, Br−, I−, CN−, and NO•. As reported with a similar metallopeptide (ref 19) the addition of over 3000 equiv of N3−, I−, Br−, and Cl− yields no change in the electronic absorption spectrum of {NiII(SODm1)}. The addition of CN− removes NiII from the metallopeptide following the addition of greater than 2 equiv of cyanide. NO• addition results in metallopeptide decomposition likely owing to S-nitrosylation. (49) We note a possible exception was recently reported where a mixed amine/amidate NiIIN2S2 system was purported to coordinate superoxide; see ref 44. To our knowledge this is the only example of exogenous ligand coordination to a square-planar mixed amine/ amidate NiIIN2S2 complex. (50) The redox potentials and S-H+ pKa vaules for NiII(SOD-Cys(6) H+) (0.610 V vs SHE; pKa = 6.2) vs NiII(SOD-Cys(6)H+)···H2O (0.571 V vs SHE; pKa = 3.5) were calculated at the PBE0/def2-tzvp level. N


Cysteinate Protonation and Water Hydrogen Bonding at the Active

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